Radioactive iodine labeling of viruses, enzymes and flourescene isothyiocyanate

ABSTRACT

An in vitro method of labeling viruses, enzymes, and fluorochrome molecules extracellularly in which a phosphate buffered saline solution is combined with a solution of KI containing I2. Next a predetermined quantity of radioactive iodine-125 is added to the solution. At this point the entity to be labeled is added, and the solution is stirred and allowed to react. A slight excess of sodium thiosulphate is added to convert any unreacted iodine to iodide, and the solution is then dialyzed.

atem [191 ie States Parikh et al.

[ Jan.7, 1975 RADIOACTIVE IODINE LABELING OF VIRUSES, ENZYMES AND FLOURESCENE ISOTHYIOCYANATE [75] Inventors: Gokaldas C. Parikh; Charles Steven Duvall, both of Brookings, S. Dak.

[73] Assignee: The United States of America as represented by the Secretary of the Navy, Washington, DC.

[22] Filed: Jan. 29, 1973 [21] Appl. No.: 327,257

[52] US. Cl. 424/1, 250/303, 252/301.1 R, 260/335, 195/103.5 R, 23/230 B [51] Int. Cl A61k 27/04 [58] Field of Search 424/194, 302; 252/301.1 R; 250/303; 260/335, 112 R;

[56] References Cited FOREIGN PATENTS OR APPLICATIONS 1,817,308 9/1970 Germany 424/1 OTHER PUBLICATIONS Quilligan, Nuclear Sci. Abs., Vol. 20, No. 24, Dec. 31, 1966, p. 5521 Item No. 45288.

Primary Examiner-Benjamin R. Padgett Attorney, Agent, or Firm-R. S. Sc iascia; P. Schneider; R. Rothman [57] ABSTRACT 12 Claims, No Drawings 1 RADIOACTIVE IODINE LABELING OF VIRUSES, ENZYMES AND F LOURESCENE ISOTHYIOCYANATE BACKGROUND OF THE INVENTION The present invention relates generally to detection of viruses, enzymes and fluorochrome molecules, and more specifically to a method of labeling these three entities with radioactive iodine to enable one to detect and count them.

At present viruses and other biologically active molecules are generally labeled by isotope incorporation inside cells, intracellularly, before detection and assaying is attempted. Such procedures require excessive amounts of time and entail high costs. Enzymes, such as RNase, are presently assayed as proteins or by their enzymatic activity. When assayed by these conventional means, the enzymes biological activity is denatured. The fluorescence of fluorochrome is presently measured by fluorometry which is difficult to quantitate by fluorescent microscopy.

SUMMARY OF THE INVENTION Both radioactive Iodine and the entity to be tagged are added to a previously prepared phosphate buffered saline solution. After allowing sufficient time for the reaction, unreacted iodine is converted to iodide, and the solution is dialyzed.

An object of the present invention is to radioactively tag materials with a minimum of equipment and in a minimum amount of time.

Another object of the invention is to extracellularly label viruses, enzymes and fluorochrome molecules in vitro.

A further object of the invention is to reduce the amount of isotope necessary to label materials.

Still another object is to allow labeling of viruses in water, food, soil, air and other mediums.

Other objects, advantages, and novel features of the present invention will become apparent from the following detailed description.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention may best be understood by reference to the following examples.

EXAMPLE I 100 ml of 0.2M phosphate buffered saline (PBS) pH 7 was prepared by combining 64 ml of stock solution A (0.15M NaCl and 0.2M Na HPO with 36 ml of stock solution B (0.15M NaCl and 0.2M NaH- PO A second solution of 0.1 N KI containing 0.0l N I was prepared by adding 0.508 grams of I to 100 ml of distilled water which was combined with another 100 ml of distilled water which 3.32 grams of KI had been added. This 200 ml solution was computed to contain 63.5 X mg. of I A third solution of 0.005 N sodium thiosulfate (Na S- 0 was made by adding 0.32 grams of Na S 0 to 250 ml of distilled water.

Three 1 ml samples of different viruses-WE, (Western Encephalitis) EHD (Epizootic Hemorrhagic) and Influenza-in which known amounts of HA (Hemagglutination), PFU (plaque forming unit) and EM (electron microscope) particles respectively were present, were prepared.

Labeling of the viruses was carried out in three mm X 25 mm screw-top, flat-bottomed vials (30 ml). 3 ml of the 0.2M PBS was added to each vial. Next 0.5 ml of the KI solution was added turning the clear solution orange. 3.6 lambdas (1 me) of carrier free, radioactive iodinewas added to each solution. Finally 1 ml of WE virus was added to the first vial, 1 ml of EHD virus was added to the second vial; and 1 ml of influenza virus was added to the third vial.

The solution was stirred with a magnetic stirring bar and allowed to react for 40 minutes.

A slight excess (enough to turn the solution from orange to clearapproximately l ml) of 0.005 N sodium thiosulphate was added to convert any unreacted iodine to iodide.

The solutions were then placed in dialysis bags and dialyzed against a 0.2M PBS solution for three days, changing the buffer every 24 hours. The dialysis bags were chosen so that any untagged material would pass through the bag leaving only tagged substances. Radiation measurements of the solutions obtained, confirmed that the viruses had been tagged.

EXAMPLE II The same three initial solutions were prepared and employed to test the efficacy of the process when employed to label enzymes. A new shipment of carrier free iodine-125 was obtained and its activity was calculated as 4.89 A/mc.

A one milliliter solution containing T1 T1 was obtained having'500,000 units. A 0.2 ml sample containing 0.2 mg or 100,000 units was employed.

In a manner similar to Example I, 3 ml of 0.2M PBS was added to a 30 ml flat-bottomed vial. Next 0.5 ml of the KI solution was added turning the clear solution orange. Next 1 me (4.89M of iodine-125 was added. The RNase was diluted with distilled water up to 1 ml and added to the solution to commence the reaction.

The solution was stirred with a magnetic stirring bar and allowed to react for 40 minutes.

A slight excess, approximately 1 ml of 0.005 N sodium thiosulphate, was added to convert any unreacted iodine to iodide.

The solution was then placed in a dialysis bag and dialyzed against a 0.2M PBS solution for three days, changing the buffer every 24 hours. After this period the solution was recovered from the dialysis bag yielding approximately 5.5 ml.

EXAMPLE III The same three initial solutions were prepared and employed to test the efficacy of the process when employed to label fluorescene, fluorescent isothiocyanate (FITC). The FITC was made up in a concentration of 4 mg/S ml of PBS. The same exact steps of Example II were carried out but instead of adding I ml of RNase, 1 ml of the FITC was added. The same stirring and dialyzation steps were completed and the dialysis bag yielded approximately 5.5 ml of solution.

Again, to analyze whether or not the samples in Examples II and [II had in fact been tagged, measurements were made with a scintillation counter yielding affirmative results. Various counters known in the art may be used for this purpose. The dialysis bags employed in all three examples were obtained from Union Carbide Corporation of New Jersey. Moreover, in all three ex amples the solution of PBS against which dialysis was accomplished was O-4C. Although carrier free iodine- 125 was employed in the present procedure, as would be apparent to those skilled in the art, other chemical forms of iodine-125 could be used to label these substances.

In addition, since the iodine-125 binds to the sulphydryl groups of the virus protein coat resulting in stable sulphenyl iodide groups, the procedure would be equally applicable to any virus having sulphydryl groups in its protein coat. In a similar manner, the iodine-125 atom covalently bonds to the sulphydryl groups of the sulphur containing amino acids-Methionine, Cystine and Cysteine-of the enzymes tested. Thus, any enzymes having sulphydryl groups in their amino acids would be equally susceptible to the labeling procedure. All viruses and enzymes of the above types and FlTC molecules can be labelled with iodine- 125 using the above process.

Obviously, many modifications and variations of the present invention arepossible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

What is claimed is: 1. A method of radioactively tagging entities comprising the steps of:

combining six parts of a 0.2M phosphate buffered saline solution having a pH of 7 with one part of a 0.1 N Kl solution containing 0.1 N

adding a predetermined quantity of carrier-free, radioactive iodine-125 to the combined solutions;

adding a predetermined quantity of a material containing the entity to be tagged to the combined solutions, said entity being from the group consisting of viruses having sulphydryl groups in their protein coat, enzymes having sulphydryl groups in their amino acids, or fluorescene isothiocyanate;

stirring the combined solutions to allow the iodine and the entity to react;

converting any unreacted iodine to iodide in the combined solutions; and

dialyzing the combined solutions to isolate the tagged entity.

2. The method of radioactively tagging entities of 5. The method of radioactively tagging entities of" r 7 claim 1 wherein the phosphate buffered saline solution consists generally of 64 ml of 0.l5M NaCl and 0.2M Na i-IP0 combined with 36 ml of 0.l5M NaCl and 0.2M NaI-l PO 6. The method of radioactively tagging entities of claim 1 wherein 1 ml of the material containing the entity to be tagged is added to the combined solution.

7. The method of radioactively tagging entities of claim 1 wherein the phosphate buffered saline solution consists generally of 64 parts of 0.5M NaCl and 0.2M Na HPO combined with 36 parts of 0.15M NaCl and 0.2M NaH PO 8. The method of radioactively tagging entities of claim 1 wherein the entity to be tagged is fluorescene isothyiocyanate.

9. The method of radioactively tagging entities of claim 1 wherein the step of converting unreacted iodine to iodide comprises:

adding a solution of sodium thiosulfate to the combined solutions.

10. The method of radioactively tagging entities of claim 1 wherein the entity to be tagged is Western Encephalitis virus.

11. The method of radioactively tagging entities of claim '1 wherein the entity to be tagged is Epizootic Hemorrhagic virus.

12. The method of radioactively tagging entities of claim 1 wherein the entity to be tagged is influenza vi- I'US. 

1. A METHOD OF RADIOACTIVELY TAGGING ENTITIES COMPRISING THE STEPS OF: COMBINING SIX PARTS OF A 0.2M PHOSPHATE BUFFERED SALINE SOLUTION HAVING A PH OF 7 WITH ONE PART OF A 0.1 N KI SOLUTION CONTAINING 0.1 N 12; ADDING A PREDETERMINED QUANTITY OF CARRIER-FREE, RADIOACTIVE IODINE-125 TO THE COMBINED SOLUTIONS; ADDING A PREDETERMINED QUANTITY OF A MATERIAL CONTAINING THE ENTITY TO BE TAGGED TO THE COMBINED SOLUTIONS, SAID ENTITY BEING FROM THE GROUP CONSISTING OF VIRUSES HAVING SULPHYDRYL GROUPS IN THEIR PROTEIN COAT, ENZYMES HAVING SULPHYDRYL GROUPS IN THEIR AMINO ACIDS, OR FLUORESCENE ISOTHIOCYANATE; STIRRING THE COMBINED SOLUTIONS TO ALLOW THE IODINE AND THE ENTITY TO REACT; CONVERTING ANY UNREACTED IODINE TO IODIDE IN THE COMBINED SOLUTIONS; AND DIALYZING THE COMBINED SOLUTIONS TO ISOLATE THE TAGGED ENTITY
 2. The method of radioactively tagging entities of claim 1 wherein the step of dialyzation comprises: placing the combined solutions into a dialysis bag; and placing the bag in a 0*-4*C 0.2M phosphate buffered saline solution having a pH of 7 for a predetermined time period.
 3. The method of radioactively tagging entities of claim 2 wherein the predetermined time period is 72 hours.
 4. The method of radioactively tagging entities of claim 3 wherein the phosphate buffered saline solution in which the bag is placed is changed every 24 hours.
 5. The method of radioactively tagging entities of claim 1 wherein the phosphate buffered saline solution consists generally of 64 ml of 0.15M NaCl and 0.2M Na2HPO4 combined with 36 ml of 0.15M NaCl and 0.2M NaH2PO4.
 6. The method of radioactively tagging entities of claim 1 wherein 1 ml of the material containing the entity to be tagged is added to the combined solution.
 7. The method of radioactively tagging entities of claim 1 wherein the phosphate buffered saline solution consists generally of 64 parts of 0.5M NaCl and 0.2M Na2HPO4 combined with 36 parts of 0.15M NaCl and 0.2M NaH2PO4.
 8. The method of radioactively tagging entities of claim 1 wherein the entity to be tagged is fluorescene isothyiocyanate.
 9. The method of radioactively tagging entities of claim 1 wherein the step of converting unreacted iodine to iodide comprises: adding a solution of sodium thiosulfate to the combined solutions.
 10. The method of radioactively tagging entities of claim 1 wherein the entity to be tagged is Western Encephalitis virus.
 11. The method of radioactively tagging entities of claim 1 wherein the entity to be tagged is Epizootic Hemorrhagic virus.
 12. The method of radioactively tagging entities of claim 1 wherein the entity to be tagged is influenza virus. 